Loss of neuronatin expression is associated with promoter hypermethylation in pituitary adenoma

The imprinted gene, neuronatin (NNAT), is one of the most abundant transcripts in the pituitary and is thought to be involved in the development and maturation of this gland. In a recent whole-genome approach, exploiting a pituitary tumour cell line, we identified hypermethylation associated loss of NNAT. In this report, we determined the expression pattern of NNAT in individual cell types of the normal gland and within each of the different pituitary adenoma subtypes. In addition, we determined associations between expression and CpG island methylation and used colony forming efficiency assays (CFE) to gain further insight into the tumour-suppressor function of this gene. Immunohistochemical (IHC) co-localization studies of normal pituitaries showed that each of the hormone secreting cells (GH, PRL, ACTH, FSH and TSH) expressed NNAT. However, 33 out of 47 adenomas comprising, 11 somatotrophinomas, 10 prolactinomas, 12 corticotrophinomas and 14 non-functioning tumours, irrespective of subtype failed to express either NNAT transcript or protein as determined by quantitative real-time RT-PCR and IHC respectively. In normal pituitaries and adenomas that expressed NNAT the promoter-associated CpG island showed characteristics of an imprinted gene where approximately 50% of molecules were densely methylated. However, in the majority of adenomas that showed loss or significantly reduced expression of NNAT, relative to normal pituitaries, the gene-associated CpG island showed significantly increased methylation. Induced expression of NNAT in transfected AtT-20 cells significantly reduced CFE. Collectively, these findings point to an important role for NNAT in the pituitary and perhaps tumour development in this gland.

An improved method for undertaking limiting dilution assays for in vitro cloning of Plasmodium falciparum parasites

Background Obtaining single parasite clones is required for many techniques in malaria research. Cloning by limiting dilution using microscopy-based assessment for parasite growth is an arduous and labor-intensive process. An alternative method for the detection of parasite growth in limiting dilution assays is using a commercial ELISA histidine-rich protein II (HRP2) detection kit. Methods Detection of parasite growth was undertaken using HRP2 ELISA and compared to thick film microscopy. An HRP2 protein standard was used to determine the detection threshold of the HRP2 ELISA assay, and a HRP2 release model was used to extrapolate the amount of parasite growth required for a positive result. Results The HRP2 ELISA was more sensitive than microscopy for detecting parasite growth. The minimum level of HRP2 protein detection of the ELISA was 0.11ng/ml. Modeling of HRP2 release determined that 2,116 parasites are required to complete a full erythrocytic cycle to produce sufficient HRP2 to be detected by the ELISA. Under standard culture conditions this number of parasites is likely to be reached between 8 to 14 days of culture. Conclusions This method provides an accurate and simple way for the detection of parasite growth in limiting dilution assays, reducing time and resources required in traditional methods. Furthermore the method uses spent culture media instead of the parasite-infected red blood cells, enabling culture to continue.

Innate transcriptional networks activated in bladder in response to uropathogenic Escherichia coli drive diverse biological pathways and rapid synthesis of IL-10 for defense against bacterial urinary tract infection

Early transcriptional activation events that occur in bladder immediately following bacterial urinary tract infection (UTI) are not well defined. In this study, we describe the whole bladder transcriptome of uropathogenic Escherichia coli (UPEC) cystitis in mice using genome-wide expression profiling to define the transcriptome of innate immune activation stemming from UPEC colonization of the bladder. Bladder RNA from female C57BL/6 mice, analyzed using 1.0 ST-Affymetrix microarrays, revealed extensive activation of diverse sets of innate immune response genes, including those that encode multiple IL-family members, receptors, metabolic regulators, MAPK activators, and lymphocyte signaling molecules. These were among 1564 genes differentially regulated at 2 h postinfection, highlighting a rapid and broad innate immune response to bladder colonization. Integrative systems-level analyses using InnateDB (http://www.innatedb.com) bioinformatics and ingenuity pathway analysis identified multiple distinct biological pathways in the bladder transcriptome with extensive involvement of lymphocyte signaling, cell cycle alterations, cytoskeletal, and metabolic changes. A key regulator of IL activity identified in the transcriptome was IL-10, which was analyzed functionally to reveal marked exacerbation of cystitis in IL-10–deficient mice. Studies of clinical UTI revealed significantly elevated urinary IL-10 in patients with UPEC cystitis, indicating a role for IL-10 in the innate response to human UTI. The whole bladder transcriptome presented in this work provides new insight into the diversity of innate factors that determine UTI on a genome-wide scale and will be valuable for further data mining. Identification of protective roles for other elements in the transcriptome will provide critical new insight into the complex cascade of events that underpin UTI.

The cytochrome P-450 isoenzyme CYP2E1 in the biological processing of industrial chemicals : consequences for occupational and environmental medicine

The importance of the isoform CYP2E1 of the human cytochrome P-450 superfamily of enzymes for occupational and environmental medicine is derived from its unique substrate spectrum that includes a number of highly important high-production chemicals, such as aliphatic and aromatic hydrocarbons, solvents and industrial monomers (i.a. alkanes, alkenes, aromatic and halogenated hydrocarbons). Many polymorphic genes, such as CYP2E1, show considerable differences in allelic distribution between different human populations. The polymorphic nature of the human CYP2E1 gene is significant for inter-individual differences in toxicity of its substrates. Since the substrate spectrum of CYP2E1 includes many compounds of basic relevance to industrial toxicology, a rationale for metabolic interactions of different CYP2E1 substrates is provided. In-depth research into the inter-individual phenotypic differences of human CYP2E1 enzyme activities was enabled by the recognition that the 6-hydroxylation of the drug chlorzoxazone is mediated by CYP2E1. Studies on CYP2E1 phenotyping have pointed to inter-individual variations in enzyme activities. There are consistent ethnic differences in CYP2E1 enzyme expression, mostly demonstrated between European and Japanese populations, which point to a major impact of genetic factors. The most frequently studied genetic polymorphisms are the restriction fragment length polymorphisms PstI/RsaI (mutant allele: CYP2E1*5B) located in the 5′-flanking region of the gene, as well as the DraI polymorphism (mutant allele: CYP2E1*6) located in intron 6. These polymorphisms are partly related, as they form the common allele designated CYP2E1*5A. Striking inter-ethnic differences between Europeans and Asians appear with respect to the frequencies of the CYP2E1*5A allele (only approximately 5% of Europeans are heterozygous, but 37% of Asians are, whilst 6% of Asians are homozygous). Available studies indicate a wide variation in human CYP2E1 expression, which are very likely based on complex gene-environment interactions. Major inter-ethnic differences are apparent on the genotyping and the phenotyping levels. Selected cases are presented where inter-ethnic variations of CYP2E1 may provide likely explanations for unexplained findings concerning industrial chemicals that are CYP2E1 substrates. Possible consequences of differential inter-individual and inter-ethnic susceptibilities are related to individual expressions of clinical symptoms of chemical toxicity, to results of biological monitoring of exposed workers, and to the interpretation of results of epidemiological or molecular-epidemiological studies.

Biological monitoring and Biological Limit Values (BLV): the strategy of the European Union

Occupational standards concerning allowable concentrations of chemical compounds in the ambient air of workplaces have been established in several countries worldwide. With the integration of the European Union (EU), there has been a need of establishing harmonised Occupational Exposure Limits (OEL). The European Commission Directive 95/320/EC of 12 July 1995 has given the tasks to a Scientific Committee for Occupational Exposure Limits (SCOEL) to propose, based on scientific data and where appropriate, occupational limit values which may include the 8-h time-weighted average (TWA), short-term limits/excursion limits (STEL) and Biological Limit Values (BLVs). In 2000, the European Union issued a list of 62 chemical substances with Occupational Exposure Limits. Of these, 25 substances received a "skin" notation, indicating that toxicologically significant amounts may be taken up via the skin. For such substances, monitoring of concentrations in ambient air may not be sufficient, and biological monitoring strategies appear of potential importance in the medical surveillance of exposed workers. Recent progress has been made with respect to formulation of a strategy related to health-based BLVs.

Biological monitoring in workers in a nitrobenzene reduction plant : Haemoglobin versus serum albumin adducts

The high priority of monitoring workers exposed to nitrobenzene is a consequence of clear findings of experimental carcinogenicity of nitrobenzene and the associated evaluations by the International Agency for Research on Cancer. Eighty male employees of a nitrobenzene reduction plant, with potential skin contact with nitrobenzene and aniline, participated in a current medical surveillance programme. Blood samples were routinely taken and analysed for aniline, 4-aminodiphenyl (4-ADP) and benzidine adducts of haemoglobin (Hb) and human serum albumin (HSA). Also, levels of methaemoglobin (Met-Hb) and of carbon monoxide haemoglobin (CO-Hb) were monitored. Effects of smoking were straightforward. Using the rank sum test of Wilcoxon, we found that very clear-cut and statistically significant smoking effects (about 3-fold increases) were apparent on CO-Hb (P = 0.00085) and on the Hb adduct of 4-ADP (P = 0.0006). The mean aniline-Hb adduct level in smokers was 1.5 times higher than in non-smokers; the significance (P = 0.05375) was close to the 5% level. The strongest correlation was evident between the Hb and HSA adducts of aniline (rs = 0.846). Less pronounced correlations (but with P values < 0.02) appeared between aniline-Hb and 4-ADP-Hb adducts (rs = 0.388), between 4-ADP and 4-ADP-HSA adducts (rs = 0.373), and between 4-ADP-Hb and aniline-HSA adducts (rs = 0.275). In view of the proposal for additional use of the aniline-HSA adduct for biological monitoring, particularly in cases of acute overexposures or poisonings, the strong correlation of the Hb and HSA conjugates is noteworthy; the ratio aniline-HSA:aniline-Hb was 1:42 for the entire cohort.

Human saliva as a tool to investigate intimate partner violence

Human saliva mirrors body’s health and well-being and many of the biomolecules present in blood or urine can also be found in salivary secretions. However, biomolecular concentrations in saliva are usually one tenth to one thousandth of the levels in blood (Pfaffe et al., 2011). Sensitive detection technology platforms are therefore required to detect biomolecules in saliva. Another road block to the advancement of salivary diagnostics is the lack of information related to healthy state saliva vs. a diseased saliva, baseline levels and reference ranges and diurnal variations. Saliva has numerous advantages over blood or urine as a diagnostic fluid: (a) the non-invasive nature of sample collection and the simple, safe, painless and cost-effective methods to collect it; (b) unskilled personnel can collect saliva samples at multiple time points; and (c) the total protein concentration is approximately a quarter of that is present in plasma, which makes it easier to investigate low abundance proteins (Pfaffe et al., 2011). Currently, saliva assays are routinely used to determine, diseases such as HIV, drugs and substances of abuse to provide information on exposure and give qualitative information on the type of illicit drug used (Kintz et al., 2009), cortisol levels for diagnosing Cushing’s syndrome (Doi et al., 2008), and use for biomonitoring of exposure to chemicals (Caporossi et al., 2010) to measure hormones (Gröschl, 2009)....

DNA methylation at the novel CpG sites in the promoter of MED15/PCQAP gene as a biomarker for head and neck cancers

Head and neck cancers (HNCs) represent a significant and ever-growing burden to the modern society, mainly due to the lack of early diagnostic methods. A significant number of HNCs is often associated with drinking, smoking, chewing beetle nut, and human papilloma virus (HPV) infections. We have analyzed DNA methylation patterns in tumor and normal tissue samples collected from head and neck squamous cell carcinoma (HNSCC) patients who were smokers. We have identified novel methylation sites in the promoter of the mediator complex subunit 15 (MED15/PCQAP) gene (encoing a co-factor important for regulation of transcription initiation for promoters of many genes), hypermethylated specifically in tumor cells. Two clusters of CpG dinucleotides methylated in tumors, but not in normal tissue from the same patients, were identified. These CpG methylation events in saliva samples were further validated in a separate cohort of HNSCC patients (who developed cancer due to smoking or HPV infections) and healthy controls using methylation-specific PCR (MSP). We used saliva as a biological medium because of its non-invasive nature, close proximity to the tumors, easiness and it is an economically viable option for large-scale screening studies. The methylation levels for the two identified CpG clusters were significantly different between the saliva samples collected from healthy controls and HNSCC individuals (Welch's t-test returning P, 0.05 and Mann-Whitney test P, 0.01 for both). The developed MSP assays also provided a good discriminative ability with AUC values of 0.70 (P, 0.01) and 0.63 (P, 0.05). The identified novel CpG methylation sites may serve as potential non-invasive biomarkers for detecting HNSCC. © the authors.

Sexual selection in true fruit flies (Diptera: Tephritidae) : transcriptome and experimental evidences for phytochemicals increasing male competitive ability

In male tephritid fruit flies of the genus Bactrocera, feeding on secondary plant compounds (sensu lato male lures = methyl eugenol, raspberry ketone and zingerone) increases male mating success. Ingested male lures alter the male pheromonal blend, normally making it more attractive to females and this is considered the primary mechanism for the enhanced mating success. However, the male lures raspberry ketone and zingerone are known, across a diverse range of other organisms, to be involved in increasing energy metabolism. If this also occurs in Bactrocera, then this may represent an additional benefit to males as courtship is metabolically expensive and lure feeding may increase a fly's short-term energy. We tested this hypothesis by performing comparative RNA-seq analysis between zingerone-fed and unfed males of Bactrocera tryoni. We also carried out behavioural assays with zingerone- and cuelure-fed males to test whether they became more active. RNA-seq analysis revealed, in zingerone-fed flies, up-regulation of 3183 genes with homologues transcripts to those known to regulate intermale aggression, pheromone synthesis, mating and accessory gland proteins, along with significant enrichment of several energy metabolic pathways and gene ontology terms. Behavioural assays show significant increases in locomotor activity, weight reduction and successful mating after mounting; all direct/indirect measures of increased activity. These results suggest that feeding on lures leads to complex physiological changes, which result in more competitive males. These results do not negate the pheromone effect, but do strongly suggest that the phytochemical-induced sexual selection is governed by both female preference and male competitive mechanisms.

Choosing an appropriate modelling framework for analysing multispecies co-culture cell biology experiments

In vitro cell biology assays play a crucial role in informing our understanding of the migratory, proliferative and invasive properties of many cell types in different biological contexts. While mono-culture assays involve the study of a population of cells composed of a single cell type, co-culture assays study a population of cells composed of multiple cell types (or subpopulations of cells). Such co-culture assays can provide more realistic insights into many biological processes including tissue repair, tissue regeneration and malignant spreading. Typically, system parameters, such as motility and proliferation rates, are estimated by calibrating a mathematical or computational model to the observed experimental data. However, parameter estimates can be highly sensitive to the choice of model and modelling framework. This observation motivates us to consider the fundamental question of how we can best choose a model to facilitate accurate parameter estimation for a particular assay. In this work we describe three mathematical models of mono-culture and co-culture assays that include different levels of spatial detail. We study various spatial summary statistics to explore if they can be used to distinguish between the suitability of each model over a range of parameter space. Our results for mono-culture experiments are promising, in that we suggest two spatial statistics that can be used to direct model choice. However, co-culture experiments are far more challenging: we show that these same spatial statistics which provide useful insight into mono-culture systems are insuffcient for co-culture systems. Therefore, we conclude that great care ought to be exercised when estimating the parameters of co-culture assays.

Development and application of Palygorskite porous ceramsite in a biological aerated filter (BAF)

Novel filter Palygorskite porous ceramsite (PC) was prepared using Palygorskite clay, poreforming material sawdust, and sodium silicate with a mass ratio of 10:2:1 after sintering at 700°C for 180 min. PC was characterized with X-ray diffraction, X-ray fluorescence, scanning electron microscopy, elemental, and porosimetry. PC had a total porosity of 67% and specific surface area of 61 m2/g. In order to assess the usefulness of PC as a medium for biological aerated filters (BAF), PC and (commercially available ceramsite) CAC were used to treat wastewater city in two laboratory-scale upflow BAFs. The results showed that the reactor containing PC was more efficient than the reactor containing CAC in terms of total organic carbon (TOC), ammonia nitrogen (NH3-N), and the removal of total nitrogen (TN) and phosphorus (P). This system was found to be more efficient at water temperatures ranging from 20 to 26°C, an air–water (A/W) ratio of 3:1, dissolved oxygen concentration >4.00 mg/L, and hydraulic retention time (HRT) ranging from 0.5 to 7 h. The interconnected porous structure produced for PC was suitable for microbial growth, and primarily protozoan and metazoan organisms were found in the biofilm. Microorganism growth also showed that, under the same submerged culture conditions, the biological mass in PC was significantly higher than in CAC (34.1 and 2.2 mg TN/g, respectively). In this way, PC media can be considered suitable for the use as a medium in novel biological aerated filters for the simultaneous removal of nitrogen and phosphorus.

Review-evaluating the molecular assays for measuring the oxidative potential of particulate matter

Several cell-free assays are currently used to quantify and detect the Reactive Oxygen Species (ROS). All of them have certain limitations, do not provide direct comparison of results and, to date, none of these assays have been acknowledged as the most suitable acellular assay and none has yet been adopted for investigation of potential PM toxicity. These assays include DTT, ascorbic acid, DCFHDA and PFN assays which have been used in measurements of the particles generated from various combustion sources such as diesel engine, wood smoke (or biomass burning) and cigarette smoke, as well as for outdoor measurements. All the probes use different units for expressing redox properties of PM. Also, their reactivity is being triggered by different types of ROS. This limits the direct comparison of the results that are reporting the toxicity of the same aerosol type measured with various probes. This study is evaluating and comparing the various assays in order to develop deeper understanding of their capabilities, selectivity as well as improve understanding of the underlying chemical mechanisms. Keywords: DTT, DCFH-DA, PFN, BPEA-nit, Ascorbic acid, oxidative potential